Integrins are a super-family of cell surface adhesion receptors which control the attachment of cells with the solid extracellular environment--both to the extracellular matrix (ECM), and to other cells. Adhesions is of fundamental importance to a cell; it provides anchorage, cues for migration, and signals for growth and differentiation. Integrins are directly involved in numerous normal and pathological conditions, and as such are primary targets for therapeutic intervention. Integrins are integral transmembrane proteins, heterodimers, whose binding specificity depends on which of some 14 .alpha.-chains is combined with which of some 8 .beta.-chains. The integrins are classified in four overlapping subfamilies, containing the .beta.1, .beta.2, .beta.3 or .alpha.v chains, and a particular cell may express several different integrins from each subfamily. In the last decade it has been shown that integrins are major receptors involved in cell adhesion, and so may be a suitable target for therapeutic intervention. Reports concerning integrins are given, for example, by E. Ruoslahti (J. Clin. Invest., 1991, 87) and R. O. Hynes (Cell, 1992, 69).
Except for erythrocytes, all human cells express one or more integrins. Their functions are regulated at many levels, but primarily, their ligand specificity depends on which chain associates with which .beta. chain in the heterodimer and on the activation state of the integrins (Hynes, 1992; Diamond and Springer, 1994). The cellular background in which the integrins operate (Chan and Hemler, 1993), and the splice-variant form of the integrin which is used (Delwel et al., 1993) may also affect specificity. Given these complexities, one of the few reliable indications of integrin specificity is to directly perturb integrin function and analyze which cellular responses are affected. The history of integrin research has shown that reagents that can specifically block integrin function are decisive factors in functional analysis, from the function blocking CSAT-antibody, which first defined an integrin .beta.1-chain (Neff et al., 1982), to the numerous vital later examples (eg. P1D6, P1B5 (Wayner and Carter, 1987), P4C10 (Carter et al., 1990), AII B2 (Hall et al., 1990), 3A3 (Turner et al., 1989), GOH3 (Sonnenberg et al., 1987), and LM609 (Cheresh and Spiro, 1987)): the field is absolutely dependent on such reagents.
The .alpha.v-series integrins are now seen to be a major subfamily, with both classical, and novel functions. As well as classically mediating cell attachment and spreading (Pytela et al., 1985; Cheresh, 1991), .alpha.v integrins have also been implicated in cell locomotion (Seftor et al., 1992), in receptor internalization (Panetti and McKeown Longo, 1993a; Panetti and McKeown Longo, 1993b), as virus co-receptors (Wickham et al., 1993), in management of the extracellular protease cascades (de Boer et al., 1993), and as regulators of tumor progression (Felding-Habermann et al., 1992). The specificities of the five known .alpha.v-series integrins, .alpha.v.beta.1 (Zhang et al., 1993), -.beta.3 (Pytela et al., 1985; Cheresh et al., 1987), -.beta.5 (Cheresh et al., 1989), -.beta.6 (Busk et al., 1992) and -.beta.8 (Moyle et al., 1991), have been partially defined, and they seem to exclusively recognize ligands bearing the RGD (-NH-arginine-glycine-aspartic acid-CO-) tripeptide sequences, including those in vitronectin (.alpha.v.beta.1, .alpha.v.beta.3, .alpha.v.beta.5), fibronectin (.alpha.v.beta.1, .alpha.v.beta.3, .alpha.v.beta.5, .alpha.v.beta.6), and von Willebrand factor, fibrinogen, and osteopontin (.alpha.v.beta.3) (e.g. 1991; Busk et al., 1992; Zhang et al., 1993; Denhardt and Guo, 1993; Smith and Cheresh, 1990;). Dimers with related specificities may be co-expressed on the same cell (eg. .alpha.v.beta.3 and .alpha.v.beta.5--for vitronectin on M21 cells) (Wayner et al., 1991), but may control independent functions. However, the overlapping ligand specificities within the .alpha.v-family itself and also between .alpha.v- and .beta.1-series integrins, means that assigning a function to a defined receptor within a particular cellular environment is problematic. Function blocking antibodies have been vital in clarifying the function of .alpha.v.beta.3 (Cheresh and Spiro, 1987; Chuntharapai et al., 1993) and .alpha.v.beta.5 (Wayner et al., 1991).
However, for the other .alpha.v-integrins, no antibodies which specify the complex and perturb function are known. In particular, few reagents which specify the .alpha.v-chain of the complex, and perturb integrin function of the whole family are available. Lehmann et al. (1994) disclosed an .alpha.v.beta.x antibody which shows no reversal of cell matrix interaction and no tumor development blocking activity.
Therefore there is intense interest in the function of .alpha.v-series integrins in tumor development. Human malignant melanoma is an increasingly prevalent aggressive skin cancer. Elevated levels of integrins .alpha.2.beta.1 (Danen et al., 1993; Etoh et al., 1992), .alpha.3.beta.1 (Natali et al., 1993; Yoshinaga et al., 1993), .alpha.4.beta.1 (Hart et al., 1991), and .alpha.6.beta.1 (Hart et al., 1991) have each been implicated in melanoma progression, but the integrins most consistently implicated are those of the .alpha.V-series. In particular, both the invasion from the primary tumor and distant metastases are characterized histologically by an increased expression of .alpha.v.beta.3 integrin, the "vitronectin receptor". Primary non-invasive tumors and non-malignant melanotic nevi express little detectable .alpha.v.beta.3, a receptor rare in healthy adult tissue (Brooks et al., 1994; Buck et al., 1990; Pignatelli et al., 1992; Lessey et al., 1992; Korhonen et al., 1991; Nesbitt et al., 1993). Immunohistochemistry of staged tumors and metastases showed a progressive increase in .alpha.V.beta.3 with invasive stage (Albelda et al., 1990; Si and Hersey, 1994), screening of melanoma lines uniformly reveals a high expression of .alpha.V-series integrins (Sanders et al., 1992; Gehlsen et al., 1992; Marshall et al., 1991), and in addition sprouting blood capillaries express .alpha.v.beta.3 during tumor angiogenesis (Brooks et al., 1994).
Studies in vivo also implicate .alpha.V.beta.3 in melanoma development. In the murine B16-F10 melanoma system, experimental lung metastasis could be suppressed by high levels of RGD-peptides (Hardan et al., 1993; Humphries et al., 1986), potent blockers of .alpha.v-integrin function. More recently, Felding-Habermann and colleagues have shown that .alpha.V-series integrins promote subcutaneous tumor growth of M21 human melanoma in immune-deficient mice. The M21 system is elegant, and consists of a suite of cells expressing different .alpha.V-series integrins (Kieffer et al., 1991; Felding-Habermann et al., 1992; Cheresh and Spiro, 1987). The parent, M21, expresses .alpha.V.beta.3 and .alpha.V.beta.5 (Wayner et al., 1991): it attaches to vitronectin and grows as a subcutaneous tumor. M21-L, a somatic variant of M21, has no detectable .alpha.V (Cheresh and Spiro, 1987): it cannot bind vitronectin and develops slow-growing tumors. M21-L4 is a transfectant of M21-L, stably re-expressing a full length .alpha.V-chain: it binds vitronectin and grows rapidly as a subcutaneous tumor (Felding-Habermann et al., 1992). Thus the presence of cell surface .alpha.V-integrins is directly correlated with M21 subcutaneous growth.
However, M21 was subjected to extreme selection pressures during the establishment of the variant lines M21-L and M21-L4. In this invention, it was found that .alpha.v-integrin function on the native M21 population can be blocked and that a surprising effect on cell behavior and tumor development can be found. Peptidic antagonists can be synthesized easily, however, their use is restricted because of their poor bio-availability, short half life in vivo, and rapid clearance from the animals (Humphries et al., 1988). Syngeneic antibodies offer an interesting alternative to peptides. They have a long half life in-vivo (Tao and Morrison, 1989; Haba et al., 1985) and their binding specificities can be well demonstrated by standard techniques. Unfortunately, although there are excellent .alpha.V-specific antibodies such as LM142 (Cheresh and Spiro, 1987), there are few that effectively block .alpha.V-integrin functions.
The specificity and biological function of the .alpha.v-family members is much debated, primarily because no reagent yet exists that will knock out the function of the whole class--there exists no potent .alpha.v blocking antibody. The revelation that the classical adhesion receptors of the integrin family support other less conventional biological functions has intensified the search for their molecular mechanisms of action. The search has revealed several unpredicted regions on the integrins that allosterically report their activation state, or, when ligated, can themselves activate integrin function. Inhibitors of integrin function, by contrast, generally occlude the active site, the classic example being RGD-peptides that replicate the integrin recognition site of many ligands of integrins of the .alpha.v-series and of 5.beta.1. Such a reagent would be very useful, not only to help elucidate these specificities and functions, but would also have potential therapeutic and diagnostic utility.